In general, the present invention relates to methods for immobilizing peptides and proteins onto a solid support.
Proteins are known to interact specifically with other molecules, such as other proteins, nucleic acids, and small-molecules, based on the three-dimensional shapes and electronic distributions of those molecules. Through these interactions, proteins are responsible for regulating a wide variety of biological processes. The discovery of compounds that interact with proteins can lead to the identification of new drug targets and the development of pharmaceutical products.
One way to search for new drug candidates is to screen large libraries of natural materials or synthesized molecules, using assay techniques that range in complexity from simple binding reactions to elaborate physiological preparations. Unfortunately, the isolation of compounds that bind specifically to biologically important molecules is difficult, in part because the selection of molecules of interest from large ensembles or repertoires is time-consuming, laborious, and costly. As advances in technology allow for the generation of larger libraries of compounds, there is a need for high throughput systems that allow for rapid and efficient screening of large numbers of compounds.
One method for screening involves the preparation of chips that include arrays of candidate molecules immobilized on a solid support. The first such arrays were nucleic acids. Proteins, however, are more difficult to immobilize and screen. It is important for proteins to have the proper orientation to ensure accessibility of their active sites to interacting molecules. Consequently, maintaining the activity of immobilized proteins at the liquid-solid interface requires different immobilization strategies than for nucleic acids.
Currently, methods for the immobilization of proteins on a support for application as screening devices involve immobilization of free proteins in solution. However, these methods are not possible with all proteins, such as when the protein is insoluble or difficult to purify. Another problem arises because covalent protein immobilization is usually carried out non-specifically by reaction of functional groups of amino acid residues throughout the protein with appropriately derivatized surfaces. This approach renders some proteins inactive due to disruption of protein structure and/or inaccessibility of the protein's binding or active sites. Even if such sites are exposed, the immobilized protein may be too close to the solid support surface to allow for interaction with reagents in the solution phase. Moreover, even though some of the immobilized proteins do retain functionality, a certain fraction of the protein sample will generally be inactive, resulting in a heterogeneous population.
Improved methods for immobilizing proteins onto a solid support are needed so that proteins exhibiting limited solubility, proteins that are difficult to purify, or proteins that are sensitive to inactivation by current immobilization techniques can be immobilized.